The leaves of Stevia rebaudiana contain a secondary metabolite called steviol, which is a member of diterpenoids. Steviol glycosides elicit a sweet taste that are up to about 300 times the sweetness of sugar, and have been used as non-caloric sweeteners in the food industry. Obesity is globally increasing as a serious social problem, and demand for non-caloric sweeteners is growing every day from viewpoints of promoting health and reducing medical expenses. Currently, Aspartame and Acesulfame Potassium, which are artificially synthesized amino acid derivatives, are used as artificial sweeteners. However, it is expected that naturally occurring non-caloric sweeteners like steviol glycosides are more likely to enjoy public acceptance.
Steviol contained in the leaves of stevia is modified with sugars finally to a glycoside called rebaudioside A with four glucose moieties attached (FIG. 1). Its precursor steviol triglycoside, stevioside, is most abundant quantitatively, and rebaudioside A and stevioside are the main components of sweetness in stevia. In addition to them, the presence of glycosides considered to be reaction intermediates and analogs with different sugars are known.
Enzyme genes encoding biosynthesis of rebaudioside A have been isolated through an expressed sequence tag (EST) analysis of stevia (Non-Patent Documents 1 and 2, Patent Document 1). Steviol is produced through hydroxylation at position 13 of ent-kaurenoic acid, i.e., a precursor of plant hormone diterpenoid, gibberellins, by cytochrome P450 enzyme ent-kaurenoic acid, 13-hydroxylase (EK13H) (FIG. 2) (Non-Patent Document 3, Patent Document 1). The 13-hydroxy group of steviol is first glycosylated (monoglucosylation) by UGT85C2 to produce steviolmonoside. The position 2 of the glucose at position 13 of steviolmonoside is further glucosylated to form steviolbioside, or the carboxyl group at position 19 of steviolmonoside is glucosylated to form a steviol diglycoside called rubusoside. Steviolbioside or rubusoside thus produced is considered to undergo further glycosylation to form steviol glycosides such as stevioside and rebaudioside A. UGT74G1 and UGT76G1 are known as enzyme genes involved in formation of steviol glycosides.
UGT74G1 is known to catalyze glucosylation of the position 19 of steviolmonoside (Non-Patent Document 1). UGT74G1 also catalyzes glucosylation of steviolbioside to produce stevioside which is a steviol triglycoside. The content of stevioside is most abundant in the leaves of stevia; stevioside is known to be approximately 250 to 300 times sweeter than sugar. This stevioside is further glucosylated by UGT76G1 to produce steviol tetraglycoside, rebaudioside A, which is the sweetest (350 to 450 times sweeter than sugar) and reportedly has a favorable quality of taste.
It is reported on steviol glycosides that addition of branched sugars especially to the glucose at position 13 results in improved quality of taste and sweetness (Non-Patent Document 4, Patent Document 2). It is thus considered that glycosidases that catalyze these reactions are important enzymes to determine sweetness qualities of stevia. 
In the previous study (Non-Patent Document 2), several types of glucosyltransferases (UGT) are reported by the EST analysis of stevia leaves. However, detailed enzyme activities of all these enzymes have not been fully investigated. Also, a homologous protein of UGT91D1 is reported only for the isolation of an truncated sequence (Patent Document 3).